The proposed research investigates novel molecular targets and processes that promise to minimize brain damage after cerebral ischemic stroke (CIS) and traumatic brain injury (TBI). Current therapeutic strategies to combat acute brain injuries have been largely unsuccessful. We discovered that the Ca2+ dependent phosphatase calcineurin (CN) can bind to PERK, a stress sensor in the endoplasmic reticulum (ER), increases its auto-phosphorylation and enhance a cellular process called the Unfolded Protein Response (UPR). The UPR attenuates protein translation during stress and gives the cell more time to recover. Significantly, our preliminary data suggest that this new protective role for CN increases cell viability after ischemic conditions in cell culture. The goal of this R21 proposal is to develop molecular interventions that can be used to specifically regulate PERK auto-phosphorylation in vivo. Ultimately, data generated from this proposal will be used to delineate the therapeutic potential of regulating the UPR during CIS and TBI. Our overall hypothesis is that, under ischemic conditions, CN directly interacts with PERK with Ca2+ as a co-factor. The formation of this protein complex promotes PERK dimerization/oligomerization and auto-phosphorylation. This, in turn, enhances inhibition of protein translation and cell viability, which reduces brain damage after injury. We have two Specific Aims: 1) Develop molecular interventions that promote, disrupt or mimic CN binding to PERK. 2) Delineate the Ca2+ dependence of CN binding to PERK in vivo. Biochemical assays and biophysical techniques will be used to map the binding interaction of CN and PERK and to generate the peptide fragments. Primary cultures of astrocytes will be used to test the efficacy of these molecular interventions in vivo. Confocal microscopy will be used to image changes in microdomains of Ca2+ near the ER. Oxygen Glucose Deprivation, an in vitro model of ischemia, will be used to determine the physiological impact of PERK auto-phosphorylation as well as our molecular interventions. If successful, the development of these peptides will serve as attractive therapeutic tools for the treatment of brain injuries.